An Optimized Transformation Protocol for Escherichia coli BW3KD with Supreme DNA Assembly Efficiency

ABSTRACT DNA cloning requires two steps: the assembly of recombinant DNA molecules and the transformation of the product into a host organism for replication. High efficiencies in both processes can increase the success rate. Recently, we developed an Escherichia coli BW3KD strain with higher transformation efficiency than commonly used cloning strains. Here, we further developed a simple method named TSS-HI (transformation storage solution optimized by Hannahan and Inoue method) for competent cell preparation, which combined the advantages of three common methods for operational simplicity and high transformation efficiency. When competent BW3KD cells were prepared using this developed method, the transformation efficiency reached up to (7.21 ± 1.85) × 109 CFU/μg DNA, which exceeded the levels of commercial chemically competent cells and homemade electrocompetent cells. BW3KD cells formed colonies within 7 h on lysogeny broth agar plates, quicker than the well-known fast-growing E. coli cloning strain Mach1. The competent cells worked effectively for the transformation of assembled DNA of 1 to 7 fragments in one step and promoted efficiencies of transformation or cloning with large plasmids. The cloning efficiency of BW3KD cells prepared by this method increased up to 828-fold over that of E. coli XL1-Blue MRF′ cells prepared by a common method. Thus, competent cells are suitable for different cloning jobs and should help with the increased demand for DNA assembly in biological studies and biotechnology. IMPORTANCE DNA transformation is commonly used in cloning; however, high transformation efficiency becomes a limiting factor in many applications, such as the construction of CRISPR and DNA libraries, the assembly of multiple fragments, and the transformation of large plasmids. We developed a new competent cell preparation method with unmatched transformation efficiency. When the BW3KD strain, derived from Escherichia coli BW25113 cells, was prepared by this method, its transformation efficiency reached up to (7.21 ± 1.85) × 109 CFU/μg DNA, which broke the record for chemically prepared competent cells. Routine cloning could be completed in 1 day due to the high growth rate of this strain. The competent cells were shown to be highly efficient for transformation or cloning with large plasmids and for the assembly of multiple fragments. The results highlight the effectiveness of the new protocol and the usefulness of the BW3KD strain as the host.

Comment on: An optimized transformation protocol for Escherichia coli BW3KD with supreme DNA assembly efficiency The authors have developed an E. coli clone, BW3KD, with a higher transformation efficiency than the commonly used cloning strains. Here, they improved the method for competent cell preparation which led to a further increase of transformation efficiency which was also superior to electrocompetent cells. A further advantage of BW3KD was that it formed colonies within 7 hours on lysogeny broth agar plates transformation worked also well with assembled DNA up to 7 fragments and also with large plasmids. The studies with BW3KD were compared with some common E. coli cloning hosts such as Mach1 or E. coli XL1-Blue MRF'.
The development of an E. coli clone with high transformation efficiency and rapid growth is extremely important in molecular biology. However, there are some questions about this paper.
Novelty: In the previous paper BW3KD has been already described and compared with its parent strain BW25113 (Yang et al. 2022, Frontiers in Microbiology). In this paper it was already described that BW3KD had a similar TE to that of BW25113. Here is another comparative description of BW3KD. Nevertheless, the already pulished work on BW3KD diminishes the novelty of the present work.
Line 61: .... one that we developed method TEDA, have been developed (5, 10). a) The sentence structure is not correct, b) Although TEDA is described in reference, it should be also briefly described here in M&M. Fig. 3. Why does increasing DNA content (volume), decrease TE? Line 168: "colonies of BW3KD could appear in less than 7 hours, but the colonies of Mach1 could not ...". How long does Mach I need?
The authors compared BW3KD with E. coli XL1-Blue MRF' and Mach1, but a also very frequent used cloning host, E. coli BL21(DE3), was not compared. Can the authors say something to BL21?
Can the authors say something about the expression of proteins, cytoplasmic and secreted? Many commercial strains are very good in this respect, not only in high TE.
For the readers and users, it would be good if at the end of the manuscript an exact transformation protocol for the competent cell preparation and transformation would be compiled.
BW3KD should be freely available to users at no cost. Also to be able to verify certain properties. This should be guaranteed by authors.
Reviewer #2 (Comments for the Author): Table 1 under "TSS-HI" "similar to TSS" as a feature is not well explained If TSS-HI is the name for this new method as in Table 1, then please put this term in the Abstract to facilitate future indexing.
Line 91, the KCM buffer is not explained. What does KCM stand for-it is explained later in methods but would be (briefly) appropriate here as well.  Figure S1: Conditions designated as optimal in Table 2 are graphed with error bars in Fig. S1. However, statistical significance of differences between treatments/groups in S1 are not indicated, although visual maxima are easy to spot generally. Some treatment differences with the optimal choices for the TSS-HI procedure indicated in Table 2 are not obviously greatly different in Fig S1. For example, S1, panel E, the chosen conditions from Table 2 show 45 s and 90s being 'equivalent' but the S1 panel E data show 30s as being pretty close to 45s/90s and it is unclear if the results are really significantly lower. Later work in the paper clearly demonstrates that the combined TSS-HI method is highly efficient, so this may not matter in how that total protocol was developed, but if significance were noted, it could help other scientists who come back to modify the TSS-HI protocol later to choose modifications to the protocol without having to see the raw data. Definition of the error bars in the Fig  S1 legend would also be needed.
Lines 155-157: It would seem appropriate to me to put suppliers for the commercial competent cells in this discussion?
Line 191: You mention TEDA earlier (line 62) but you did not define it there (other than as a method which is cited), nor here. At line 62 it is probably not necessary to define if it is just a name and the acronym is not important. However, in line 191 those who don't know what the TEDA mixture is may be confused. This should be better explained or spelled out. I think this entire paragraph could deal with some more explanation as the TEDA method is new (2018) and even though it has made a big splash in terms of assembly utility for the cost involved, there are likely many who don't know about it yet. I'd like to see more explanation here.
Line 194: the Pkat-eGFP fragment cloning mentioned should also be better explained-what is the fragment, what is being cloned, etc.
Line 219: I feel like a new section header would be appropriate here to indicate that the work discussed in this paragraph goes beyond the paragraph before.
Line 238: Another section header here is also helpful and appropriate. Line 314-315: This sentence is a bit unclear and could be explained better.
Line 363-364: Given this is a methods paper, I would love to see a supplemental method or supplemental table that gives an easy to use recipe format for how to make the TSS-HI buffer. While perhaps less traditional in a scientific paper, the ability for scientists to find and use this recipe would be of great interest to the community and be a valuable resource.
Staff Comments:

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Review of "An optimized transformation protocol for Escherichia coli BW3KD with supreme DNA assembly efficiency" by Yang et al.
In this paper, the authors report a strain, BW3KD, of E. coli, with increased transformation efficiency compared to common cloning strains. High efficiency was maintained with large constructs and with cloning projects involving up to 7 fragments. The protocol to make these competent cells is also reported.
The conclusions of the authors appear to be supported by the data presented. This work is important and could result in benefit to the scientific community by producing higher efficiency competent cells for lower cost, and this will enable transformation of difficult substrates/assemblies as well.
I recommend publication of this work. I do have some specific concerns, noted below. Table 1 under "TSS-HI" "similar to TSS" as a feature is not well explained If TSS-HI is the name for this new method as in Table 1, then please put this term in the Abstract to facilitate future indexing.
Line 91, the KCM buffer is not explained. What does KCM stand for-it is explained later in methods but would be (briefly) appropriate here as well.  Figure S1: Conditions designated as optimal in Table 2 are graphed with error bars in Fig. S1. However, statistical significance of differences between treatments/groups in S1 are not indicated, although visual maxima are easy to spot generally. Some treatment differences with the optimal choices for the TSS-HI procedure indicated in Table 2 are not obviously greatly different in Fig S1. For example, S1, panel E, the chosen conditions from Table 2 show 45 s and 90s being 'equivalent' but the S1 panel E data show 30s as being pretty close to 45s/90s and it is unclear if the results are really significantly lower. Later work in the paper clearly demonstrates that the combined TSS-HI method is highly efficient, so this may not matter in how that total protocol was developed, but if significance were noted, it could help other scientists who come back to modify the TSS-HI protocol later to choose modifications to the protocol without having to see the raw data. Definition of the error bars in the Fig S1 legend would also be needed. Line 219: I feel like a new section header would be appropriate here to indicate that the work discussed in this paragraph goes beyond the paragraph before.
Line 238: Another section header here is also helpful and appropriate.
Line 314-315: This sentence is a bit unclear and could be explained better.
Line 363-364: Given this is a methods paper, I would love to see a supplemental method or supplemental table that gives an easy to use recipe format for how to make the TSS-HI buffer.
While perhaps less traditional in a scientific paper, the ability for scientists to find and use this recipe would be of great interest to the community and be a valuable resource.

Point-by-point responses to reviewers
Reviewer #1 (Comments for the Author): Comment on: An optimized transformation protocol for Escherichia coli BW3KD with supreme DNA assembly efficiency The authors have developed an E. coli clone, BW3KD, with a higher transformation efficiency than the commonly used cloning strains. Here, they improved the method for competent cell preparation which led to a further increase in transformation efficiency which was also superior to electrocompetent cells. A further advantage of BW3KD was that it formed colonies within 7 hours on lysogeny broth agar plates transformation worked also well with assembled DNA up to 7 fragments and also with large plasmids. The studies with BW3KD were compared with some common E. coli cloning hosts such as Mach1 or E. coli XL1-Blue MRF'.
The development of an E. coli clone with high transformation efficiency and rapid growth is extremely important in molecular biology. However, there are some questions about this paper.
Novelty: In the previous paper BW3KD has been already described and compared with its parent strain BW25113 (Yang et al. 2022, Frontiers in Microbiology). In this paper, it was already described that BW3KD had a similar TE to that of BW25113. Here is another comparative description of BW3KD. Nevertheless, the already published work on BW3KD diminishes the novelty of the present work.

Response:
The previous paper focuses on the recognition of BW25113 as an efficient host for DNA assembly. Various mutants were created to understand why it was efficient. BW3KD with the deletion of three genes was not responsible. The key gene was identified as recA, which is normally inactivated in the cloning strains to promote the stability of the cloned genes. This manuscript describes a new method to prepare competent cells with improved TE and showed that BW3KD is suitable for handling large plasmids for efficient DNA assembly. We believe that BW3KD can be a useful host for DNA assembly.
1. Line 61: .... one that we developed method TEDA, have been developed (5, 10). a) The sentence structure is not correct, b) Although TEDA is described in reference, it should be also briefly described here in M&M.
Response: a) The sentence was changed to "Several simplified Gibson methods, including the TEDA (T5 exonuclease-dependent assembly) method, have been developed" (Line 61-62). b) The brief procedure of TEDA was added in M&M (Line387-396).

Fig. 3. Why does increasing DNA content (volume), decrease TE?
Response: When the number of clones was counted by the unit of DNA, it declined (Fig.  3A). We speculated that there are two reasons. First, when a high amount of TEDA-treated DNA was mixed with cells, multiple assembled DNA molecules could enter one cell. Hence, the transformation efficiency per unit of DNA decreased. Second, the presence of certain ions in the TEDA reaction buffer may reduce the TE of competent cells. The TE of TSS-HI depends on the types of ions and their strengths in the TSS-HI buffer and KCM buffer (Fig. 1A&B). We speculated that excess ions from the TEDA buffer would weaken the TE of competent cells prepared by TSS-HI.
3. Line 168: "colonies of BW3KD could appear in less than 7 hours, but the colonies of Mach1 could not ...". How long does Mach I need?
Response: 10 hours were required for Mach1. That is 2-3 hours more than that required for BW3KD. The information was added in the text (Line 173).
4. The authors compared BW3KD with E. coli XL1-Blue MRF' and Mach1, but an also very frequently used cloning host, E. coli BL21(DE3), was not compared. Can the authors say something to BL21?
Response: E. coli BW25113 belongs to the K-12 lineage, and BL21 is of the strain B lineage. BL21(DE3) is commonly used to overexpress cloned genes due to the lack of Lon and OmpT proteases (Jeong H, et al.; Genome Announc. 2015 Mar 19;3(2):e00134-15.). BL21(DE3) commonly has low transformation efficiency (TE). The TE of commercial competent BL21(DE3) was normally kept between 1*10 6 ~ 5*10 7 CFU/μg DNA (Agilent, Thermo, NEB). When the competent cells of BL21(DE3) were prepared by the TSS-HI method, its TE was 2.1*10 7 CFU/μg DNA. It is much lower than XL1-Blue MRF' and Mach 1 (Fig. 2A). For cell growth, our results showed that BL21(DE3) grew faster than XL1-Blue MRF' but similar to Mach1 (Fig. 1 listed in this response letter). Response: According to the purpose of use, commercial strains are divided into clonal and protein-expression types. Cloning-type strains generally harbor special characteristics for DNA cloning, but rarely have special properties for protein expression (Refer to the links at the end of this paragraph). Apart from high TE, the other main advantages include easy cloning of large plasmids, T1 phage resistance, easy plasmid preparation, the rapid growth of cells, etc. Our results have proved that BW3KD prepared by TSS-HI not only has high TE but also could clone large plasmids (Fig. 5A&B). In addition, it has the characteristic of rapid growth (Fig. 2E). The knockout of endA1 has improved the quality of plasmid extraction, and the knockout of the fluA gene makes this strain could tolerate T1 phage. Therefore, the BW3KD strain already has comparative advantages in many respects compared with the commercial cloning strains.
Commonly used commercial protein-expression strains generally require the T7 expression system to achieve protein overexpression and purification. Although  Table 2 are graphed with error bars in Fig. S1. However, the statistical significance of differences between treatments/groups in S1 is not indicated, although visual maxima are easy to spot generally. Some treatment differences with the optimal choices for the TSS-HI procedure indicated in Table 2 are not greatly different in Fig S1. For example, in S1, panel E, the chosen conditions from Table 2 show 45 s and 90s being 'equivalent' but the S1 panel E data show 30s as being pretty close to 45s/90s and it is unclear if the results are significantly lower. Later work in the paper demonstrates that the combined TSS-HI method is highly efficient, so this may not matter in how that total protocol was developed, but if significance were noted, it could help other scientists who come back to modify the TSS-HI protocol later to choose modifications to the protocol without having to see the raw data. A definition of the error bars in the Fig S1 legend would also be needed.

Response:
Yes. Statistical significance of differences is important. The statistical significance of differences was added in Fig S1. 5. Lines 155-157: It would seem appropriate to me to put suppliers for the commercial competent cells in this discussion?

Response:
The catalog numbers, the brands, and their origins for the commercially competent cells were indicated in the text. (Line 158 -Line 160) 6. Line 191: You mention TEDA earlier (line 62) but you did not define it there (other than as a method that is cited), nor here. In line 62 it is probably not necessary to define if it is just a name and the acronym is not important. However, in line 191 those who don't know what the TEDA mixture is may be confused. This should be better explained or spelled out. I think this entire paragraph could deal with some more explanation as the TEDA method is new (2018) and even though it has made a big splash in terms of assembly utility for the cost involved, there are likely many who don't know about it yet. I'd like to see more explanation here.
Response: Thanks for your suggestion. A detailed explanation for TEDA was added to the text. The full name of TEDA was provided in Line 62. The detailed procedure of TEDA was added in the section on materials and methods (Line 387 -Line 394). 7. Line 194: the Pkat-eGFP fragment cloning mentioned should also be better explained-what is the fragment, what is being cloned, etc.

Response:
The explanation was given in Line 195-198. In addition, a more detailed explanation was given in the section on materials and methods (Line 400 -Line 402).
8. Line 219: I feel like a new section header would be appropriate here to indicate that the work discussed in this paragraph goes beyond the paragraph before.
Response: It was added "Competent BW3KD cells prepared with TSS-HI facilitated multi-fragments assembly" in Line 222 as a section header.
Line 238: Another section header here is also helpful and appropriate.
Response: It was added "Competent BW3KD cells prepared with TSS-HI facilitated large DNA transformation and cloning" in Line 243 as a section header.
9. Line 314-315: This sentence is a bit unclear and could be explained better.

Response:
The sentence was changed to "Because the TE is high enough, direct transformation of untreated DNA fragments can meet the needs for simple cloning (Fig. 3)." in Line 320-321.
10. Line 363-364: Given this is a methods paper, I would love to see a supplemental method or supplemental table that gives an easy-to-use recipe format for how to make the TSS-HI buffer. While perhaps less traditional in a scientific paper, the ability for scientists to find and use this recipe would be of great interest to the community and be a valuable resource. It would be nice to put your responses to the reviewer no.1 's comments (no. 4 and 5) to the revised version of the manuscript, as these are the points that should be informed to the readers of Microbiology spectrum as well. As for the response to comment no. 7, please specify what is xx.
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